More is known about the neuromuscular synapse than any other synapse. Its geometrical simplicity and experimental accessibility have provided us with a relatively detailed account of what a synapse looks like and how a synapse functions. We remain, however, rather poorly informed about the mechanisms involved in the formation and maintenance of this highly specialized and organized region of cell-to-cell contract. An understanding of how neuromuscular synapses form both during development and during regeneration will not only provide important information about the steps and mechanisms of synapse formation in the peripheral nervous system, but also about the presently less tractable analysis of synapse formation in the central nervous system. This proposal is concerned with further characterization of two proteins, a 43 kD and a 300 kD protein, that are components of the postsynaptic membrane at nerve-muscle synapses. The function of these proteins is unknown, but their location at the synapse suggests that they may have a role in the formation, maturation and/or maintenance of the synapse. The experiments described in this proposal are designed to identify cellular components that are necessary and sufficient to target the 43 kD protein to the postsynaptic membrane. The proposed experiments are also designed to determine whether the 43 kD protein is required for clustering of AChRs and synapse formation. We will use Xenopus oocytes as a system to reconstitute targeting of the 43 kD protein, we will investigate methods for disrupting genes encoding synaptic proteins in C2 muscle cells by homologous recombination and continue experiments designed to prevent translation of RNA encoding synaptic proteins by injection of anti-sense RNA into developing Xenopus embryos. In addition, experiments described in this proposal are designed to characterize further the 300 kD subsynaptic protein.